Transcriptome Based Reference Genes Selection And The Expression Analyses Of Genes Involved In The Biosynthesis Of Three Metabolic Pathways

Moringa oleifera Lam., belonging to a single-genus family Moringaceae, is a fast-growing perennial plant. This tree species contains rich nutrition and medical components, and thus has great economic value, especially on medicine, oil and forage. In this study, high-quality transcriptome data was built, a certain number of candidate reference genes were selected, genes involved in the biosynthesis of flavonoid and terpenoid were chosen and their expression patterns in various tissues were compared, genes related to the biosynthesis of fatty acids were selected and their expression pattern in pods during different developmental stages were also analyzed. The main research results are listed as followed:1, 38,672,496 clean reads and 45,052 unigenes were obtained through transcriptome sequencing, which enriched the transcriptome data base.2, 18 candidate reference genes were selected, including GAPDH, PEPC, ACP1, ACP2, UBCE, TUA1, TUA2, RPL1, RPL2, MDH1, MDH2, ACT, UEP, EF1, EF2, TUB, CYP1, CYP2.3, RT-q PCR technique and three algorithms(ge Norm, Norm Finder, Bestkeeper) were used to analyze and evaluate the expression stability of 18 candidate reference genes under 6 different experimental conditions, including young pods in different developmental stages, various tissues and 4 different stress treatments(chilling, high temperature, Na Cl and PEG). The result indicated that genes RPL1 and ACP2 were the most suitable reference genes in all tested samples. And the most stable reference genes would change in different experimental conditions.And the most stably expressed reference genes under Na Cl and PEG treatments analyzed by ge Norm were used for the expression normalization of three stress-related genes(Cu/Zn-SOD, Fe-SOD and Mn-SOD). The expression levels of these three genes increased with the time, verifying the feasibility of the selected reference genes.4, Several genes involved in the biosynthesis of flavonoid were selected, including PAL, 4CL, CHS, F3 H, DFR and LAR. RT-q PCR was used to quantify their expression in various tissues(root, stem, mature leaves, young leaves, young pods). Our results showed that these keys genes generally had higher expression levels in root and leaves, and the root and leaf may be the main organ which synthesize the flavonoid.5, Terpenoid biosynthesis-related genes(HMGR, DXS, DXR, GPPS and CPR) were screened, and their expression pattern in different organs(root, stem, mature leaves, young leaves, young pods) were analyzed. Results demonstrated that M.oleifera may also hold the MEP-led terpenoid biosynthesis pathway, and mature leaf was the main organ for synthesizing terpenoid.6, Genes involved in fatty acids biosynthesis were picked, including ACCase, FAD2, KASI, SAD, FATA, FATB and LACS. The quantitative analysis in pods revealed that the synthesized fatty acids by young pods might generally be used for the development of yound seeds. And the expression levels of the genes related to unsaturated fatty acids made no big difference in pods during different developmental stages, but may show function during the stage of maturity.